This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. ABSTRACT: Knowledge gained from our progress on developing a "macroscopic" mixer/sprayer device over the past two years has been applied to developing a microfluidic version of this technology (described fully below). The macroscopic mixer/sprayer continues to be useful for testing new approaches for improving the methodology (e.g. testing of new grid supports and treatments to improve hydrophilicity) as it is more robust for routine operation. Also, this mixer/sprayer is useful for experiments that do not require short time intervals (e.g >200 milliseconds). A manuscript describing this work is currently being written. A second technique for performing time resolved cryo-electron microscopy involves flashing grids containing photo-reactive caged reactants as they are being plunged into cryogen. We have finalized our implementation of this technology and Dr. Tapu Shaikh wrote up the work.